How does the inheritance of chloroplast and mitochondrial DNA differ from nuclear DNA?

C. Chloroplast DNA

Chloroplast DNA (cpDNA) in photosynthetic land plants is also a circular genome, which varies in size from about 120,000 to 247,000 nucleotides, largely because of a large inverted repeat that includes genes for the rRNA subunits. Each chloroplast contains from about 22 to 900 cpDNA copies and each encodes 123 genes. These include four genes for rRNA, 20 genes for ribosomal proteins, 30 genes for tRNAs, many of the photosynthetic proteins, six of the nine genes for ATPase, and chloroplast RNA polymerase. Unlike mtDNA, 15 of the cpDNA genes contain introns. Most chloroplast proteins are encoded by the nucleus, chloroplast ribosomes consist of 52 proteins but only 19 of them are encoded by plastid genome.

cpDNA is transmitted maternally in most flowering plants, biparentally in a few, and paternally in gymnosperms. cpDNA genes have been shown to transpose to the nucleus and there is good evidence that mtDNA, cpDNA, and nuclear genomes exchange genes. The rate of cpDNA evolution generally appears slow both in terms of primary nucleotide sequence and in terms of gene rearrangement. Because of the large size of the cpDNA genome, most systematic treatments have involved restriction site or sequence determinations for particular genes or have monitored the taxonomic distributions of unique cpDNA structural features across higher-level plant taxa. Nonetheless, some studies have uncovered considerable intraspecific cpDNA variation as well, and this suggests that portions of the cpDNA should be useful in population genetics. Again, several primers for amplifying cpDNA genes in a number of plant species are available (e.g., ndhF: Olmstead and Sweere, 1994; rbcL: Palumbi, 1996).

A. Changes in gene order

Chlamydomonas cpDNA is marked by gene loss, numerous rearrangements, and in some species a dramatic increase in SDRs, with respect to those of land plants (Figure 24.3). The dynamic evolution of Chlamydomonas cpDNAs was first demonstrated in a series of reports by Claude Lemieux and colleagues (Lemieux and Lemieux, 1985; B. Lemieux et al., 1985; C. Lemieux et al., 1980, 1985). Although chloroplast gene content is conserved among Chlamydomonas species, gene order is not, as shown with DNA gel blot experiments in C. reinhardtii, C. eugametos, and C. moewusii (B. Lemieux et al., 1985; Boudreau et al., 1994). Changes in gene order appear to have resulted mostly from inversions and a limited number of deletions. Chromosome reconstruction models predict stepwise rearrangements that account for the differences in cpDNAs between Chlamydomonas species. For example, a three-step model with one deletion and two inversions can account for the differences between C. moewusii and C. pitschmannii (Boudreau and Turmel, 1995), while a ten-step model with one deletion and nine inversions can account for differences between C. gelatinosa and C. reinhardtii (Boudreau and Turmel, 1996). Between C. reinhardtii and Chlorella, a minimum of 72 inversions can explain the differences in gene order (de Cambiaire et al., 2007). Genomic rearrangements correlate with the abundance and locations of repeat sequences, and species with large numbers of SDRs tend to have more rearrangements (Maul et al., 2002; Pombert et al., 2005; Cui et al., 2006).

Cloning of Plant Genes in E. coli Vectors

Except for chloroplast DNA genes (see e.g. Coen and others, 1977), relatively few plant genes have thus far been cloned in E. coli. Recently cDNA copies of some plant storage proteins have been cloned, such as, the two subunits of the maize zein protein (Burr, 1978: Wienand, Brüschke, and Feix, 1978). Sidloi-Lumbroso and Schulman (1977) have purified the soybean leghemoglobin mRNA. One can therefore expect that cDNA copies - and possibly genes - of leghemoglobin will soon be available in E. coli vectors.

Shared Coding and DNA Editing

The transfer of genes from cpDNA to the nucleus is an ongoing evolutionary process. A similar process is also observed in mitochondria. Complete loss of redundant and extraneous genes seems to occur easily in close symbioses, perhaps because a streamlining of the genome confers a replicative advantage. In addition, intracellular horizontal transfer of genes may occur, facilitated by the proximity of the chloroplast, mitochondrial, and nuclear genes. The direction of transfer is strongly biased toward the nucleus, although chloroplast-to-mitochondria transfers are also observed. A result of horizontal transfer is a shared coding for some essential chloroplast structures including the ribosomes. For example, the large subunit of the ribulose-1,5-bisphosphate carboxylase oxygenase gene (Rubisco; rbcL) is encoded in the plastid, whereas the small subunit (rbcS) is encoded in the nucleus in higher plants, green algae, and glaucophytes. This makes the relationships even more obligate among the chloroplast, mitochondrial, and nuclear genomes of plant cells. Comparisons of cpDNA in phylogenetically widely separated plants revealed a parallel gene loss in independent lineages for many genes. Genes with regulatory functions were preferentially transferred from chloroplast to the nucleus. Thus, the regulation of gene activities in plants is strongly dominated by the nuclear genome. Transfer to the nucleus is also observed but occurred less frequent for genes involved in translation and photosynthesis. The transfer of genes from chloroplasts to the nucleus required the evolution of complex mechanisms to ensure the transport of gene products into chloroplasts. The evolutionary advantages of biparental inheritance and recombination are suspected to be the main driving forces for the transfer of genes from chloroplasts to the nucleus and the development of transport systems of enzymes from the cytoplasm to chloroplasts.

RNA editing results in a RNA sequence deviating from its DNA template. Chloroplast RNA editing, in particular, conversions from C to U and, less frequently, from U to C, is mainly observed in land plants and is particularly common in hornworts and ferns, but has also been observed in seed plants. The process of RNA editing in plants seems to be similar for mitochondrial and chloroplast genes. RNA editing is essential for the expression of different proteins since it not only results in the change of amino acid sequences but also in the change of start or stop codons. Several genes and gene families involved in RNA editing have been identified, but the importance of the process for the evolution of plants is still unknown.

7.8.5 Isopropylmalate dehydrogenase

The gene encoding this enzyme has been isolated from Brassica napus chloroplast DNA by complementation of a yeast leu 2 mutant. The cDNA encodes a 52 kDa protein which has a putative chloroplast transit peptide. Analysis of the sequence of the isopropylmalate dehydrogenase protein, suggested that the enzyme is more similar to bacterial than fungal proteins (Ellerstrom et al., 1992). The gene encoding the enzyme has also been isolated from potato (Jackson et al., 1993).

A preliminary report has indicated that the final three enzymes of leucine synthesis can be detected in spinach chloroplast extracts (Hagelstein et al., 1993).

C. Methylation of chloroplast DNA

Sager and her colleagues proposed that a restriction-modification system is responsible for the selective degradation of chloroplast DNA from the minus parent in crosses of Chlamydomonas (Sager and Lane, 1972; Sager and Ramanis, 1973, 1974; Sager and Kitchin 1975). They subsequently published several papers suggesting that this system is based on protection of chloroplast DNA of plus gametes by methylation, consistent with a shift in buoyant density observed in plus gametes and in young zygotes (Sager and Lane, 1972). Burton et al. (1979) reported that chloroplast DNA from plus gametes contained a measurable quantity of 5-methyl cytosine, whereas no methylation was detected in chloroplast DNA from minus gametes or from vegetative cells of either mating type. These results were confirmed by Sano et al. (1980) using antibodies specific for 5-methyl cytosine. In zygotes assayed 6 hours after mating, the plus chloroplast DNA appeared to be heavily methylated, and some methylation was also seen in the minus chloroplast DNA, which was largely degraded by this time (Burton et al., 1979; Royer and Sager, 1979). Feng and Chiang (1984) reported that during gametogenesis methylation of deoxycytidine increased at least 20-fold over the level seen in vegetative cells. Although methylation increased in both plus and minus cells, it was always at least threefold higher in plus. In fully differentiated plus gametes, 12.1% of the deoxycytidine residues were methylated, and within 7 hours after zygote fusion this level had risen to nearly 50% (Feng and Chiang, 1984).

Arguing against the hypothesis that methylation protected chloroplast DNA of the plus parent from degradation, a very high level of constitutive methylation resulting from the nuclear me1 mutation did not alter the pattern of chloroplast DNA inheritance (Bolen et al., 1982; see also Dyer, 1982). However additional methylation of chloroplast DNA was observed in plus cells of this mutant (Sager and Grabowy, 1983). Feng and Chiang (1984) reported normal uniparental inheritance in crosses where methylation was inhibited by treatment of gametes with l-ethionine or 5-azacytidine (Feng and Chiang, 1984), although Umen and Goodenough (2001b) reported that 5-aza-2-deoxycytidine did alter the inheritance pattern. Taken together, these results suggested that hypermethylation per se did not protect plus DNA from degradation but that certain specific sites must be methylated if this mechanism is to work. Also, these specific sites would have to be methylated even in the presence of inhibitors of methylation, as in the experiments by Feng and Chiang (1984). Dedifferentiation of gametes by restoration of nitrogen to the culture medium led to gradual loss of methylation at a rate consistent with dilution of methyltransferase activity by cell division (Sano et al., 1984). No rapid loss of methylation was observed, as might have been expected if an enzymatic demethylating activity were present.

A mechanism that absolutely eliminated chloroplast DNA from minus gametes would of course result in 100% UP+ inheritance. In Sager's early experiments, this was very nearly the case, with only about 0.1% of zygotes showing BP or UP− inheritance (Sager and Ramanis, 1963, 1967). In experiments in the Boynton-Gillham laboratory, however, up to 5% of zygotes expressed markers from the minus parent (Gillham, 1969; Gillham et al., 1974), and there is evidence that even more zygotes may harbor “hidden” copies of the chloroplast genome contributed by the minus parent (see below). The discrepancy in exceptional zygote frequencies seen in the two laboratories was attributed to differences in the procedures by which gam-etes were formed, although strain differences may also have played a role (Sears et al., 1980). To account for the existence of exceptional zygotes in terms of a restriction-modification mechanism, one must assume either that some chloroplast DNA molecules in minus gametes are protected, or that the plus restriction system is somewhat inefficient. Another observation that should be taken into account is the demonstration by Wurtz et al. (1977) that reduction of the number of copies of chloroplast DNA in plus cells by treatment with the thymidine analog 5-fluorodeoxyuridine (Figure 7.4) leads to an increase in the number of BP and UP− zygotes. In contrast to UV treatment, which produces mostly BP rather than UP− zygotes at sublethal doses, 5-fluorodeoxyruridine treatment even at the lowest effective doses yields a substantial fraction of UP− zygotes. Some UP+ zygotes are always seen even at higher doses, however, consistent with other lines of evidence suggesting that total elimination of chloroplast DNA is lethal (Liu et al., 1993; Boudreau et al., 1997a).

The presence of DNA methyltransferase activity specific to gametic and zygotic cells was documented by Sano et al. (1981), and a chloroplast-specific enzyme was later characterized (Nishiyama et al., 2002, 2004). Contrary to the prediction that specific sites needed to be methylated to protect chloroplast DNA, this enzyme appeared to be nonselective; that is, methylation of all cytosine residues occurred, regardless of the neighboring sequence of the DNA. When minus cells were transformed with the DNA encoding this enzyme, under control of a constitutive promoter, the same nonselective pattern of methylation was observed. When these transgenic cells were crossed to normal plus cells, the frequency of UP− and BP zygotes increased substantially. Nishiyama et al. (2004) concluded that their results did support a restriction-modification mechanism for protection of chloroplast DNA.

However, the results of Umen and Goodenough (2001b) and Nishimura et al. (2002) suggest that the explanation is more complex. Umen and Goodenough found that in plus cells treated with the methylation inhibitor 5-aza-2-deoxycytidine, the hypomethylated DNA was not degraded in the young zygote, as a simple restriction-modification model would predict, but that it persisted until zygote germination, at which point it failed to replicate at normal levels. They proposed that germination is the critical time at which unmethylated or damaged chloroplast DNA is finally destroyed.

The nuclease characterized by Nishimura et al. (2002) degrades minus chloroplast DNA to the nucleotide level, prior to chloroplast fusion, rather than leaving small DNA fragments as would be expected for a restriction endonuclease. Nishimura et al. considered several possibilities for how plus chloroplast DNA might be protected from the nuclease, especially in view of the evidence indicating that methylation, either general or site-specific, is probably not sufficient to explain all the results. Since the nuclease requires Ca2+ for full activation, they postulated that regulation of calcium levels within the chloroplast is important in the protection process, and called attention to the growing number of papers on zygote-specific gene expression. In particular, the EZY1 gene encodes a protein that localizes to chloroplast nucleoids immediately after mating (Armbrust et al., 1993) and is therefore a candidate for involvement in the mechanism of protection. Taking into consideration the results of Umen and Goodenough (2001b) showing higher replication rates for methylated plus DNA in germinating zygotes, Nishimura et al. suggested that two or more distinct mechanisms might operate cooperatively to bring about uniparental inheritance of chloroplast genes.

Inference of Gene Flow in Limber Pine

The organellar genomes of pines are ideal for measuring gene flow, as mtDNA has maternal inheritance and chloroplast DNA (cpDNA) has paternal inheritance in pines. These different modes of inheritance allow us to explicitly identify gene flow mediated by pollen and by seeds. In addition, pollen and seeds have disparate potentials for dispersal. The wind-borne pollen have the potential to travel great distances, but in contrast, the seeds of pines usually fall within a circle that has a radius equal to the height of the tree.

Limber pine, Pinus flexilis (Figure 2), is native to western North America, where it is primarily restricted to windy ridges and scree slopes from the Sierra Madre of Mexico to the Canadian Rockies, from Mt. Pinos in southern California to the Black Hills of South Dakota. The seeds of limber pine are dispersed and planted by Clark’s nutcracker, Nucifraga columbiana. The bird and pine are engaged in a mutualism sculpted by evolution. Limber pine relies on the bird to harvest, disperse, and plant its seeds. Clark’s nutcracker relies on limber pine seeds to get through the winter. Both the bird and the pine have evolved morphological traits (a sublingual pouch, wingless seeds) to better serve and exploit their partner. The birds usually cache seeds on windy or south-facing slopes that will be free of snow in winter, and this explains the curious distribution of limber pine. A bird can carry ~30 limber pine seeds in its sublingual pouch. When its pouch is full, the bird flies to a propitious site for caching and harvesting seed. The flight distances are highly variable; although the record flight exceeds 20 km, most flights are very short, a few meters to a few hundred meters.

The potentials for dispersal of pollen and seed lead biologists to expect high gene flow in genes dispersed by pollen (nuclear genes, cpDNA) and low gene flow for genes dispersed solely by seed. This hypothesis was tested with a study of gene flow among populations of limber pine in the Front Range of Colorado. The populations were distributed from tree line at the Continental Divide to an isolated stand of trees 100 miles to the east, on an escarpment on the Great Plains. Haplotype frequencies were used to calculate Fst for both cpDNA and mtDNA, and gene flow was inferred from Fst with the equation directly above. The Fst values were 0.02 and 0.68 for cpDNA and mtDNA, respectively, suggesting that the number of migrants among populations per year are 12.25 for pollen and 0.12 for seeds. The gene flow of cpDNA is high, and should tend to homogenize the frequencies of cpDNA haplotypes and nuclear genes among populations within distances of ~100 miles. In contrast, the gene flow of mtDNA is below the threshold at which the influence of genetic drift predominates. So mtDNA is expected to vary more among populations than nuclear genes and cpDNA, and genetic drift will cause populations to diverge with respect to mtDNA haplotypes.

4.1 Number and frequency of NUMTs and NUPTs in different organisms

In addition to the transfer of functional genes from mitochondria and plastids to the nucleus, nonfunctional mtDNA and cpDNA segments are also found in the nuclear genome and are referred to as NUMTs (Lopez et al., 1994) and NUPTs (Timmis et al., 2004), respectively. Following the pioneering work that detected mtDNA in nuclear DNA by hybridization with mtDNA probes (Dubuy and Riley, 1967), genome sequencing made it possible to identify NUMTs and NUPTs in yeast and plants by bioinformatic methods (Blanchard and Schmidt, 1995, 1996). With the increasing availability of whole genome sequences, NUMTs and NUPTs are being discovered in an ever widening spectrum of eukaryotes, ranging from protists to mammals (Hazkani-Covo et al., 2010; Richly and Leister, 2004a,b). One of the first genomewide surveys encompassed 13 nuclear genomes (Richly and Leister, 2004a). By 2010, a set of 72 new eukaryotic (nuclear and organelle) genome sequences had become available, allowing the NUMT and NUPT repertoire to be surveyed in 85 fully sequenced genomes, including those of 20 fungi, 11 protists, 7 plant/moss/algae, and 47 animals (Hazkani-Covo et al., 2010). With the advent of advanced sequencing methods, the inventory of known NUMTs and NUPTs is continuously expanding, and recent reports include analyses of norgDNA in the demosponge Amphimedon queenslandica, a representative of the oldest phyletic lineage of animals (Erpenbeck et al., 2011), Equus caballus, the domestic horse (Nergadze et al., 2010), the jewel wasp Nasonia vitripennis (Viljakainen et al., 2010), maize (Roark et al., 2010), and rice (Akbarova et al., 2011). Depending on the search strategy, the level of genome completion, and changes in the curation of the available genome sequence data, the number of orgDNAs detected in a given nuclear genome can vary (Hazkani-Covo et al., 2010). For example, with the inclusion of 4.7 Mb of heterochromatic sequence that was unavailable in the previous versions of the D. melanogaster genome, the NUMT content has increased from 0.5 kb (Richly and Leister, 2004a) to the current value of 10.3 kb (Hazkani-Covo et al., 2010). Irrespective of the aforementioned methodologically based variations, the NUMT and NUPT contents are highly variable among species. The abundance of NUMTs in eukaryotic genomes ranges from undetectable (e.g., in Anopheles gambiae; Richly and Leister, 2004a) to more than 800 kb in the rice genome and 2.1 Mb in the opossum Monodelphis domestica (Hazkani-Covo et al., 2010). Similarly, the abundance of NUPTs in nuclear genomes varies from undetectable (in the green alga Ostreococcus sp. RCC809, two apicomplexans and the stramenophile Aureococcus anophagefferens) to more than 1 Mb in O. sativa subsp. japonica (Smith et al., 2011). The fraction of the nuclear genome represented by NUMTs or NUPTs is usually less than 0.1% (Leister, 2005), but it can be higher in flowering plants (Hazkani-Covo et al., 2010; Richly and Leister, 2004a,b) and some fungi (Hazkani-Covo et al., 2010). Because mutation and deletion rapidly make NUMT and NUPT sequences unrecognizable as such, 0.1% represents the steady-state level of recently incorporated orgDNA at any given point in time (Hazkani-Covo et al., 2010). NUMTs and NUPTs vary not only in overall length but also in their size and frequency distributions (Hazkani-Covo et al., 2010; Richly and Leister, 2004a,b; Smith et al., 2011). For example, the largest insertions of norgDNA reported so far are a 620-kb partially duplicated insertion of the 367-kb mtDNA in A. thaliana (Stupar et al., 2001) and a 131-kb NUPT in rice (Huang et al., 2005; The Rice Chromosome 10 Sequencing Consortium, 2003). In terms of NUMT frequency, Apis mellifera, the Western honeybee, is among the leaders, with more than 1000 orgDNA insertions in its nuclear genome (Behura, 2007; Pamilo et al., 2007), although these are relatively short, with an average length of 96 bp. The highest frequencies of NUPTs are found in V. vinifera and Glycine max, which have more than 3000 nuclear genome insertions each with an average length of around 200 bp (Smith et al., 2011).

2. Replication of chloroplast DNA

In early experiments with 15N–14N density shifts, Chiang and Sueoka (1967a) had reported that most chloroplast DNA replication occurred during the light period and that replication was semiconservative. Catto and Le Gal (1972) similarly reported a peak of thymidine incorporation into relatively AT-rich DNA between 7 and 8 hours into the light period. Experiments by Lee and Jones (1973) indicated that in fact the density shift was gradual and that synthesis did not occur in a single burst. Subsequent work by Chiang and his colleagues (Chiang, 1971; Grant et al., 1978) showed that some incorporation of radioactive precursors occurred in both the light and the dark periods, but suggested that the incorporation in the dark period was the result of DNA repair rather than new synthesis. However, Turmel et al. (1980, 1981) found that net accumulation of chloroplast DNA occurred throughout the cell cycle with maximum synthesis taking place in the dark period, coincident with chloroplast division and cytokinesis.

The extent of dispersive labeling observed in their density transfer experiments led Turmel et al. (1981) to postulate that chloroplast DNA molecules engage in repeated heteroduplex formation whereby homologous single-stranded segments are exchanged. This process would also account for chloroplast DNA recombination in vegetative cells (see Chapter 7). Analyses of chloroplast DNA in crosses in which the parental DNAs are distinguishable by restriction site and length polymorphisms are consistent with this model: recombination appears to involve discrete, continuous segments of chloroplast DNA, rather than multiple interchanges between a single pair of genomes (Lemieux et al., 1984a, b; Newman et al., 1992).

Two replicative origins were identified in a single 0.42-kb region of the chloroplast genome by Wang et al. (1984) and were localized by electron microscopy of replicating molecules by Waddell et al. (1984). These were further characterized by Wu and colleagues (Wu et al., 1986a, b; Hsieh et al., 1991; Chang and Wu, 2000; see also Volume 2, Chapter 24). Chlamydomonas chloroplast sequences that promote autonomous replication in yeast were also identified (Loppes and Denis, 1983; Vallet et al., 1984; Houba and Loppes, 1985), and are distinct from the origins of replication (Vallet and Rochaix, 1985). Additional recombination-dependent origins were identified by Woelfle et al. (1993).

Two distinct chloroplast DNA polymerase activities were identified in Chlamydomonas (Keller and Ho, 1981; Wang et al., 1991), but were not been fully characterized in functional terms (see Volume 2, Chapter 24). Other proteins that have been identified include a DNA primase (Nie and Wu, 1999), and topoisomerases (Thompson and Mosig, 1985; Woelfle et al., 1993).

(ii) Structure of the chloroplast genome

The DNA of the chloroplast is double stranded, circular and ranges in size from 120 to 169 kb (Fig. 1.25). Chloroplast DNA from Pisum sativum is approximately 40 μm in circumference. Covalent, closed and superhelical chloroplast DNA of around 40–45 μm have been found in all higher plants examined to date.

Pea chloroplast DNA contains approximately 18 ribonucleotides. This evidence is primarily based on the fact that (1) there are alkali-labile sites in the closed circular DNA. Alkali-sensitivity is a characteristic feature of ribonucleotide linkages (2) the closed circular chloroplast genome was converted to open circular DNA upon treatment with pancreatic RNase and RNase T1. The significance of these ribonucleotides is unknown, although they may function as specific recognition sites. Chloroplasts and mitochondrial DNA can exist in multiple circular forms of dimers and catenated monomers. These forms represent about 3 and 1.5%, respectively, of the genome in higher plants. These dimers appear to have been formed from the fusion of two circular monomers.

One of the noticeable features of the chloroplast genome is the presence of the large inverted repeats (IR). The inverted repeats are separated by a large and small single copy region (LSC and SSC, respectively). In the liverwort, Marchantia polymorpha, the small single copy region contains 19 813 bp and the large single copy region contains 81 095 bp. The pea and broad bean chloroplast genomes are devoid of these inverted repeats. The chloroplast genome contains all the chloroplast rRNA genes (3–5 genes), tRNA genes (about 30 genes) and all the genes for the ‘housekeeping’ proteins synthesized in the chloroplast (100–150 genes). About one fifth of the tobacco chloroplast genome is transcribed and the majority of RNA formed is believed to represent mRNA and not tRNA or rRNA. Major proteins synthesized in chloroplasts include a soluble large subunit-ribulose bisphosphate carboxylase and a 32 kDa membrane protein associated with photosystem II. A number of unassigned open reading frames have been identified in the plastid genome. Most of the genome encodes proteins involved in plastid transcription and translation or in photosynthesis. Therefore, the great majority of the plastid genome tends to be expressed in photosynthetically active cells. Interestingly, all plastid genomes contain the gene that encodes the respiratory chain NADH dehydrogenase which is similar to that found in human mitochondria. This gene is expressed highly in the plastid and may point to the existence of a respiratory chain in chloroplasts.

There are no histones associated with the chloroplast DNA and there is also no 5-methylcytosine which is a characteristic feature of nuclear DNA. The DNA is believed to be attached to the chloroplast membranes and also extends into the stroma. The complete chloroplast genome from liverwort and tobacco has been sequenced (Fig. 1.25). Each chloroplast contains a single DNA molecule present in multiple copies. The number of copies varies between species; however, the pea chloroplasts from mature leaves normally contain about 14 copies of the genome. There can be in excess of 200 copies of the genome per chloroplast in very young leaves.

The chloroplasts of photosynthetic plant cells are believed to have arisen from a symbiotic association between a photosynthetic bacterium and a non-photosynthetic eukaryotic organism. The eukaryotic host cell presumably engulfed the endosymbiotic organism by endocytosis, and this would have accounted for the double membrane of the present day chloroplast. At one point the endosymbiotic organism must have had a genome that encoded all the proteins required for independent existence. Perhaps many of the genes were already present within the nuclear genome of the host cell. The gene encoding the small subunit of ribulose bisphosphate carboxylase may have been present in the endosymbiont.